Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced profiling of protein phosphorylation

DOI Web Site 11 References
  • Kinoshita-Kikuta Emiko
    Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University
  • Kinoshita Eiji
    Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University
  • Koike Tohru
    Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University

Bibliographic Information

Other Title
  • 今後の展望１　中性条件で泳動を行う改良型　Ｐｈｏｓ－ｔａｇ　ＳＤＳ－ＰＡＧＥを用いたリン酸化タンパク質解析
  • 中性条件で泳動を行う改良型Phos-tag SDS-PAGE を用いたリン酸化タンパク質解析
  • チュウセイ ジョウケン デ エイドウ オ オコナウ カイリョウガタ Phos-tag SDS-PAGE オ モチイタ リン サンカ タンパクシツ カイセキ

Search this article

Abstract

We describe an improved Phos-tag SDS-PAGE (Zn2+-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) to detect shifts in the mobility of phosphorylated proteins. Our alternative technique (Mn2+-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn2+-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements and utilities were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant substrate protein tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole cell lysates. Additionally, the Zn2+-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn2+-Phos-tag SDS-PAGE gels. We can thus present a simple, convenient, and more reliable "in-house" gel system for phosphate-affinity SDS-PAGE.<br>

Journal

References(11)*help

See more

Related Projects

See more

Keywords

Details 詳細情報について

Report a problem

Back to top